The long-term goal of this proposal is to understand the normal biological function of the hMDM2 oncoprotein and how its overexpression can induce tumorigenesis. The short-term objective is to test the hypothesis that hMDM2 induces cell cycle arrest, possibly by interacting with the cell cycle regulatory proteins. Inactivation of this function is one of the causes of tumorigenesis. The working hypotheses are based on the exciting observations that mMDM2 induces GO/G1 arrest in normal human diploid cells. Elimination of the growth arrest function enhances the tumorigenic potential of NIH3T3 cells. Furthermore, hMDM2 associates with two cell cycle regulatory proteins, cyclin E and p34 (cdc2). This proposal has four specific aims. 1) Consequence of hMDM2 overexpression in normal breast epithelial cells and breast tumor- derived cells will be analyzed. The domain(s) of hMDM2 needed to confer these growth alterations will be determined using deletion mutants of the oncoprotein. Identification of these domains will enable us to detect interactions of hMDM2 with other proteins (such as cyclin E) important for cellular growth regulator functions. 2) Different splice variants of mdm2 mRNA will be isolated from an hMDM2 overexpressing breast cancer cell line MCF-7 and a murine tumorigenic cell line 3T3DM (harbors amplified mdm2 gene). The growth regulatory properties of the proteins encoded by the different splice variants will be analyzed. 3) The consequence of hMDM2-cyclin E interaction on the cyclin E-associated kinase -activity and on cell growth will be determined. 4) Since hMDM2 associates with P34 (cdc2), how hMDM2 modulates the activation of p34 (cdc2) kinase activity and how this interaction modulates cell growth will be determined. Completion of these specific aims will elucidate a novel growth regulatory function of hMDM2 and how perturbation of that function leads to tumorigenesis.